Two Signal Amplification Strategies For Improving The Detection Sensitive Of Conventional Immunoassay

Enzyme-linked immunosorbent assay（ELISA）and immunochromatographic assay（ICA）are the two most common immunoassay informats and have been widely used for detecting various target analytes,including disease biomarkers,small molecules and metal ions.Conventional ELISA usually applied horseradish peroxidase（HRP）to catalyze the tetramethyl benzidine（TMB）for the generation of colored molecules as signal reporters,whereas conventional ICA depends on 30-40nm gold nanoparticles（AuNPs）as as signal probes.However,these two methods suffer from relatively low detection sensitivity ranging fromμg/mL to ng/mL owing to their relatively weak signal output,largely limiting their applification in trace target detection.Therefore,to improve the detection sensitivities of these two approaches is significant for broadening their practical application.In this work,two new signal amplification strategies were developed to enhance the detection sensitivity of traditional ELISA and ICA,respectively.The detailed contents are as follows:（1）Increasing the signal molecules load of probe is one of the common strategies to improve the detection sensitivity of immunoassay.Nanomaterials have been extensively employed as carriers for improving the enzyme loading due to their large specific surface area,further contributing to amplifying the detection signal of ELISA for achieving the ultrasensitive target detection.Here,we first used a thiolated amino/carboxyl-ligand modified multi-branched gold nanoflower（AuNFs）as skeleton to encapsulate iron porphyrins（FeTPPCl）and Fluorescein（AuNF@FeTPPCl and AuNF@Fluorescein）as alternatives to horseradish peroxidase（HRP）.Through the combination of the HRP mimic enzyme activity of FeTPPCl and Fluorescein with the ultrahigh loading capacity of AuNFs,AuNF@FeTPPCl and AuNF@Fluorescein were used as enzyme labels for constructing a direct competitive ELISA for highly sensitive detection of fumonisin B₁（FB₁）in food and a sandwich ELISA for ultrasensitive analysis of alpha fetoprotein（AFP）in serum,respectively.Meanwhile,the fluorescence emission of Fluorescein was further used as signal output to fabricate a dual-mode“colorimetric-fluorescence”ELISA method for AFP,greatly improving the accuracy of traditional single mode immunoassay.To this end,a thiolated amino ligand consistsing of hydrophobic alkane chain,a tetra（ethylene glycol）unit,and a functional terminal amino group was first synthesized and then modified onto 90 nm AuNF surface to form hydrophobic pocket via the Au-S bond.Later,the FeTPPCl and Fluorescein molecules were encapsulated into the hydrophobic pocket on the AuNF surface to produce two different nanozymes of AuNF@FeTPPCl and AuNF@Fluorescein through the hydrophobic interaction,respectively.The synthesized AuNF@FeTPPCl and AuNF@Fluorescein exhibited ultrahigh loading level with 2.4×10⁶ FeTPPCl and 3.67×10⁶ Fluorescein molecules per AuNF.Further analysis for enzyme activity showed that the encapsulation and release of FeTPPCl and Fluorescein did not significantly decrease the catalytic activity,while the catalytic constants（Kcat）of these two nanozymes for H₂O₂ and TMB are ca.1 to2 orders magnitidue higher than native HRP,which is beneficial for increasing the detection sensitivity of conventional ELISA.In addition,these nanoenzymes also exhibited stronger environmental tolerance and long-term preservation stability,greatly improving the reproducibility and stability.Based on the above analysis,on the one hand,the resultant AuNF@FeTPPCl was selected as carrier for the conjugation of BSA and FB₁ for preparing the competition antigen,and then used to establish a highly sensitivie direct competitive ELISA.Under the optimal experimental conditions,the calibration curve for FB₁ quantitative detection was constructed with the regression equation of y=-15.59ln（x）+123.84（R²=0.9954）.According to this equation,the half-maximal inhibitory concentration(IC₅₀)was calculated as 101 pg/mL,which is ca.250-fold lower than that of conventional HRP-based ELISA.We also evaluated the reliability of the competitive ELISA for FB₁ detection by blindly analyzing fifteen of FB₁-spiked real corn,rice,and wheat samples containing known concentrations of FB₁,and the average recoveries rangeed from 89.3%to 114%with a coefficient variation（CV）of<20%,indicating that the AuNF@FeTPPCl-based competitive ELISA was well-suitable for FB₁detection in real samples.On the other hand,the AuNF@Fluorescein was chosen as enzyme labels to develop a dual-mode sandwich ELISA for sensitive and accurate determination of AFP in serum.Under the optimal experimental conditions,using the colorimetric signal as output,the linear regression equation was represented as y=2.0785x+109.8 with a limit of detection（LOD）of 17.7 pg/mL,15-fold lower than that of conventional ELISA,whereas using the fluoresce signal as output,the linear regression equation was described as y=1.2924x+0.1117 with a LOD of 29 fg/mL,9300-fold lower than that of conventional ELISA.The reliability of the developed dual-mode sandwich ELISA was further estimated via a correlation analysis with Abcam chemiluminescence immunoassay kit by simultaneously analyzing 20clinical serum samples with these two methods,and results indicated that the two methods well agreed with each other with high correlation coefficients of 0.986 and0.997 when used the colorimetric and fluorescent signal output,respectively.Furthe sepecificity analysis of our proposed dual-mode immunoassay incidcated that no obvious cross-reactions against other several common serum protein biomarkers were observed,demonstrating an excellent selectivity for AFP determination.These findings suggested that the proposed dual-mode ELISA was highly suitable for ultrasensitive and reliable measurement of AFP in serum.（2）Increasing the signal strength of AuNP probes is an important strategy to improve the sensitivity of AuNP-ICA.Traditional Au or Ag signal enhancement depends on the reduction deposition of Au or Ag nanoshells on the surface of AuNP probes for remarkably increased particle size to achieve color signal amplification,thereby greatly enhancing the detection sensitivity of ICA.However,conventional reduction deposition growth of Au or Ag nanoshells on the AuNP surface suffers from the irregular and uncontrollable growth,thus causing poor stability and reproducibility and limiting its practical application.To solve the above problems,here we applied polyethyleneimine（PEI）as as template skeleton for the coordination of copper ions(Cu²⁺)to realize the controllable growth of Cu nanoshell on the AuNP surface for largely magnifying the colorimetric signal intensity of AuNP,further increasing the detection sensitivity of ICA.In this case,the Cu²⁺ions can bind with the AuNP probes captured on the test lines of strip under the assistance of PEI and then the adsorbed Cu²⁺ions on the AuNP surface was reduced into Cu atom by sodium ascorbate.Later,the reduced Cu nanoshell was deposited on the AuNP surface for increased AuNP size to significantly enhance the color signal strength.Under the optimized conditions,the amplified AuNP-ICA exhibited ultrahigh sensitivity for E.coil O157:H7 with a LOD of 6 CFU/mL in milk within 20 minutes,which are ca.three orders of magnitude higher than traditional AuNP-ICA without signal amplification.Further selectivity evaluation of the developed method was conducted through detecting 17 common food-borne pathogens and results indicated excellent discrimination ability against other non-target bacteria.The robustness and reliability of the improved AuNP-ICA method were estimated by analyzing 15 E.coil O157:H7-spiked milk samples and the average recoveries ranged from 84%to116%with CV lower than 12%.In general,the proposed method can be applied for rapid,on-site and ultrasensitive quantitative detection of E.coil O157:H7 in milk.